Abstract
Glutamyl-tRNA (Glu-tRNAGlu) is the common substrate for both protein translation and heme biosynthesis via the C5 pathway. Under normal conditions, an adequate supply of this aminoacyl-tRNA is available to both pathways. However, under certain circumstances, Glu-tRNAGlu can become scarce, resulting in competition between the two pathways for this aminoacyl-tRNA. In Acidithiobacillus ferrooxidans, glutamyl-tRNA synthetase 1 (GluRS1) is the main enzyme that synthesizes Glu-tRNAGlu. Previous studies have shown that GluRS1 is inactivated in vitro by hydrogen peroxide (H2O2). This raises the question as to whether H2O2 negatively affects in vivo GluRS1 activity in A. ferrooxidans and whether Glu-tRNAGlu distribution between the heme and protein biosynthesis processes may be affected by these conditions. To address this issue, we measured GluRS1 activity. We determined that GluRS1 is inactivated when cells are exposed to H2O2, with a concomitant reduction in intracellular heme level. The effects of H2O2 on the activity of purified glutamyl-tRNA reductase (GluTR), the key enzyme for heme biosynthesis, and on the elongation factor Tu (EF-Tu) were also measured. While exposing purified GluTR, the first enzyme of heme biosynthesis, to H2O2 resulted in its inactivation, the binding of glutamyl-tRNA to EF-Tu was not affected. Taken together, these data suggest that in A. ferrooxidans, the flow of glutamyl-tRNA is diverted from heme biosynthesis towards protein synthesis under oxidative stress conditions.
Highlights
Heme is a fundamental molecule for living organisms, as the cofactor for several proteins and enzymes involved in cellular processes, such as transport of gases, redox reactions and electron transport [1,2]
Given that glutamyl-tRNA synthetase 1 (GluRS1) is inactivated by H2O2 in vitro [13], we sought to determine whether GluRS1 activity is decreased in vivo in A. ferrooxidans exposed to H2O2
Cellular extracts from A. ferrooxidans treated with 1 mM H2O2 were obtained, and the specific activity of GluRS1 was evaluated in these extracts by the aminoacylation of tRNA2Glu, a specific substrate of GluRS1 [9,11]
Summary
Heme is a fundamental molecule for living organisms, as the cofactor for several proteins and enzymes involved in cellular processes, such as transport of gases, redox reactions and electron transport [1,2]. In chemolithoautotrophic bacteria like Acidithiobacillus ferrooxidans that use the C5 pathway to synthesize tetrapyrroles, high demand for Glu-tRNAGlu for heme biosynthesis is expected, due to the high cytochrome content required for respiration using poor electron donors, such as ferrous ions [9,10]. This bacterium has a complex system of glutamyl-tRNA formation composed of two non-discriminating glutamyl-tRNA synthetases (GluRS1 and GluRS2) and up to four tRNAGlu isoacceptors, with GluRS1 serving as the main enzyme for Glu-tRNAGlu formation [9,11,12].
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